PRODUCT INFORMATION SHEET
Gateway™ LR Clonase™ II Enzyme Mix
Catalog Number 11791-020 and 11791-100
Doc. Part No. 11791.II.pps Pub. No. MAN0001032 Rev. A.0
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Product description
Invitrogen™ Gateway™ LR Clonase™ II enzyme mix is a proprietary enzyme and buffer
formulation containing the bacteriophage lambda recombination proteins Integrase
(Int) and Excisionase (Xis), the E. coli-encoded protein Integration Host Factor (IHF),
and reaction buffer provided in a single mix for convenient reaction set up. Gateway™
LR Clonase™ II enzyme mix catalyzes in vitro recombination between an entry clone
(attL-flanked “gene”) and an attR-containing destination vector to generate an attBcontaining expression clone. Store Gateway™ LR Clonase™ II enzyme mix at –20°C
(non-frost-free freezer) for up to 6 months. For long-term storage, store at –80°C.
Gateway™ technology
Gateway™ Technology is a universal cloning method that takes advantage of the sitespecific recombination properties of bacteriophage lambda to provide a rapid and
highly efficient way to move DNA sequences into multiple vector systems. The
Gateway™ Technology is schematically represented below.
The attB × attP reaction is mediated by Gateway™ BP Clonase™ II enzyme mix; the
attL × attR reaction is mediated by Gateway™ LR Clonase™ II enzyme mix. ccdB is the F
plasmid-encoded gene that inhibits growth of E. coli and “gene” represents any DNA
segment of interest (e.g. PCR product, cDNA, genomic DNA).
For Research Use Only. Not for use in diagnostic procedures.
Contents and storage
Cat. no.
11791-020
(20 rxns)
Cat. no.
11791-100
(100 rxns)
Gateway™ LR Clonase™ II Enzyme Mix
40 μL
200 μL
Proteinase K Solution (2 μg/μL)
40 μL
200 μL
pENTR™-gus Positive Control (50 ng/μL)
20 μL
20 μL
Components
Storage
conditions
Store at –20°C
(non-frost-free
freezer)
Important guidelines
• pENTR™-gus is provided for use as a positive control in the LR reaction and is an
entry clone containing the Arabidopsis thaliana β-glucuronidase (gus) gene. Refer to
our website (thermofisher.com) for a map and sequence of pENTR™-gus.
• We recommend using plasmid DNA purified with the PureLink™ HQ Mini Plasmid
Purification Kit (Cat. no. K2100-01). Mini-prep (alkaline lysis) DNA preparations are
adequate for Gateway™ cloning reactions; however, in general, such DNA cannot be
quantitated by UV absorbance due to contaminating RNA and nucleotides. Estimate
concentrations by gel electrophoresis in comparison with standard DNA (e.g. DNA
Mass Ladder, Cat. no. 10068-013 or 10496-016).
• For LR recombination reactions, the most efficient substrates are supercoiled attLcontaining entry vectors and supercoiled attR-containing destination vectors. For
large (>10 kb) entry clones or destination vectors, linearizing the entry clone or
destination vector may increase the efficiency by up to 2-fold.
• To increase the number of colonies containing the desired expression clone, increase
the incubation time from the recommended 1 hour to 2 hours-overnight. Longer
incubations are recommended for plasmids ³10 kb to increase the yield of colonies.
• We recommend using 50–150 ng entry clone per 10 μL reaction. Highest colony
yields are typically obtained using 150 ng entry clone and 150 ng destination vector.
Do not use >150 ng entry clone as you may obtain colonies containing multiple DNA
molecules (often with an associated “small colony” phenotype). Using <50 ng entry
clone will generate fewer colonies.
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Gateway™ LR Clonase™ II Enzyme Mix Product Information Sheet
Methods
Prepare LR Reaction
LR Clonase™ II enzyme mix is supplied as a 5X solution. If you wish to scale the
reaction volume, make sure the LR Clonase™ II enzyme mix is at a final concentration
of 1X. For a positive control, use 100 ng (2 μL) of pENTR™-gus.
1. Add the following components to a 1.5-mL microcentrifuge tube at room
temperature and mix:
• 1–7 μL entry clone (50–150 ng)
• 1 μL destination vector (150 ng/μL)
• TE buffer, pH 8.0, to 8 μL
2. Thaw on ice the LR Clonase™ II enzyme mix for about 2 minutes. Vortex the LR
Clonase™ II enzyme mix briefly twice (2 seconds each time).
3. To each sample (step 1), add 2 μL of LR Clonase™ II enzyme mix to the reaction
and mix well by vortexing briefly twice. Microcentrifuge briefly.
4. Return LR Clonase™ II enzyme mix to –20°C or –80°C storage.
5. Incubate reactions at 25°C for 1 hour.
6. Add 1 μL of the Proteinase K solution to each sample to terminate the reaction.
Vortex briefly. Incubate samples at 37°C for 10 minutes.
Transform
1. Transform 1 μL of each LR reaction into 50 μL of One Shot™ OmniMAX™ 2 T1
Phage-Resistant Cells (Cat. no. C8540-03). Incubate on ice for 30 minutes. Heatshock cells by incubating at 42°C for 30 seconds. Add 250 μL of S.O.C. Medium
and incubate at 37°C for 1 hour with shaking. Plate 20 μL and 100 μL of each
transformation onto selective plates
2.
Note: Any competent cells with a transformation efficiency of >1.0 × 108
transformants/μg may be used.
Transform 1 μL of pUC19 DNA (10 ng/mL) into 50 μL of One Shot™ OmniMAX™
2 T1 Phage-Resistant Cells as described above. Plate 20 μL and 100 μL on LB
plates containing 100 μg/mL ampicillin.
Expected results
An efficient LR recombination reaction will produce >5000 colonies if the entire LR
reaction is transformed and plated.
Gateway™ LR Clonase™ II Enzyme Mix Product Information Sheet
3
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The information in this guide is subject to change without notice.
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30 October 2015